The overarching goal of this R21 proposal, A Controllable Microfluidic Gradient Device for Studying Neuronal Polarization, is to demonstrate and explore the capabilities of a novel microfluidic concentration-gradient generator, as it relates to developmental and regenerative neurobiology. The device, consisting of a series of alternating cell-culture chambers and reagent channels that are interconnected via micro-channels, establishes and maintains steady concentration gradients within a static cell-culture chamber for neuronal culture. Concentration gradients of both small molecules (pharmaceutical agents and second messengers) and macromolecules (neurotrophins and other proteins) are easily achieved and quantified in this device. Thus, quantitative studies of neuronal polarization and axon pathfinding of neurons in response to micro- environmental cues can be accomplished. Three Specific Aims will be pursued. Specific Aim 1 involves optimizing the design of the proposed microfluidic device, as well as its fabrication and measurement. Specific Aims 2 and 3 focuses on evaluating four hypotheses of neuronal polarization and axon pathfinding that are not currently quantifiable using current protocols. In particular, Specific Aim 2 involves assessing growth and guidance of embryonic rat hippocampal neurons in response to defined micro-environmental cues (BDNF and a membrane-permeable cAMP analogue) established in the device. Specific Aim 3 involves determining if localized concentration gradients of known guidance cues (BDNF and Sema3A) can induce axon specification in embryonic Xenopus spinal cord neurons. The concentration gradient requirements needed for long-range axon guidance will be quantitatively assessed. The proposed work, if successful, will have significant impact in developmental neurobiology by providing the first quantitative description of mammalian polarization and axon guidance. Because multiple guidance cues work in concert to regulate these developmental processes in vivo, it would be imperative for future studies to examine quantitatively the interplay of these environmental signals. Because this is a highly-interdisciplinary collaboration involving microfluidics, bioengineering, and neurobiology, this R21 is relevant to the missions of both NINDS and NIBIB. The proposed R21 team consists of Lydia L. Sohn (PI), Associate Professor of Mechanical Engineering at University of California, Berkeley, Sarah Heilshorn (co-PI), Assistant Professor of Materials Science & Engineering at Stanford University, and Mu-ming Poo (consultant), Professor and Division Head of Neurobiology in the Dept. of Molecular & Cellular Biology at University of California, Berkeley. Principal Investigator/Program Director (Last, First, Middle): Sohn, Lydia Lee Project Narrative The overall relevance of this R21 proposal, A Controllable Microfluidic Gradient Device for Studying Neuronal Polarization, is to provide a new quantitative technology based on microfluidics that will enable the understanding of developmental and regenerative neurobiology. It will have a high impact in clinical regeneration and guidance of injured axons. PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page Continuation Format Page [unreadable] [unreadable]